Fig. 4

Additional approaches to confirm the detection of aggregated mouse tau in primary neuronal cultures by application of artificial K18-tau fibrils, proximity ligation assay (PLA) and tau-immunodepleted AD5 sample. A Primary hippocampal neurons were treated with a K18-tau fibrils in concentrations of 0.1 and 1 µM at 7DIV and 18DIV, inoculated for 14 days (14PID) and for 3 days (3PID), respectively. Cultures were fixed with 4% PFA and 1% Triton-X100 to extract soluble tau. Compared to controls (cells treated with medium only), the K18-tau fibrils-inoculated cultures show the presence of insoluble mouse tau in an inoculum dose-dependent manner. In b neurons were treated at 7DIV with 1 µM of K-18-tau monomers or fibrils and fixed with 4% PFA and 1% Triton-X100 at 21DIV. In K18-tau fibrils treated neurons, insoluble tau colocalized with AT8-positive staining in projections (yellow arrows). Pathologically insoluble tau (AT8-positive) is also observed around nucleus in more diffuse manner (orange arrowheads). Compared to K18-tau monomers’ treatment, the presence of K18-tau fibrils contributes to conversion of endogenous mouse tau into more pathological tau species. B In situ proximity ligation assay (PLA). a Schematic representation of PLA experiment for detection of aggregated mouse tau. In mouse tau aggregates, tau molecules are in proximity and can be captured by mouse-specific tau antibodies bound to the N-terminal part of tau. These antibodies are then recognized by secondary anti-IgG PLUS/MINUS PLA probes, which are conjugated with short complementary oligonucleotide sequences with a fluorophore. The oligonucleotides undergo ligation and amplification in situ to increase the sensitivity of the fluorescent signal. b PLA images of mouse tau in ice-cold methanol fixated cultures (21DIV) inoculated for 14 days with two different concentrations (15 and 45 ng) of AD5 samples demonstrate that fluorescent signal (green) is specific to the occurrence of aggregated mouse tau. Controls of full PLA staining of AD3-inoculated wells with no cells and AD3-inoculated cell cultures following the PLA staining protocol without ligase confirm that PLA signal is specific to aggregated mouse tau. c One-way ANOVA was applied to number of particles per nuclei from six images per treatment and showed statistical significance (*** p < 0.001, n = 6), and Bonferroni test also confirmed the inoculum concentration-dependent aggregation of mouse tau (medium vs. 15 ng: *p = 0.0235, medium vs. 45 ng: ***p = 0.0001, 15 ng vs. 45 ng of AD5-tau, *p = 0.0219). C Immunodepleted AD5-tau sample as a control. a Western blot: AD5 tau-immunodepleted (ID) of sarkosyl-insoluble human tau by combined AT8 and Tau5 antibody immunomagnetic separation shows a significant decrease of HMW tau and lack of AT8 tau compared to the original AD5 sample. b Primary neurons treated with combined-ID AD5 sample show no aggregated mouse tau after 14 days of inoculation whereas aggregated mouse tau signal is detected in the cultures treated with the original AD5-tau sample. The images are presented as maximum intensities of 35 z-stacks (0.35 µm each), scale bars: 50 µm of large images, 20 µm of cropped areas. The staining for aggregated tau was performed after ice-cold methanol fixation